以纤维蛋白凝胶为载体培养兔角膜缘上皮细胞

Cultivation of corneal epithelial cells using fibrin gel

目的研究兔角膜缘上皮细胞(corneal limbus epithelialcell,CLEC)在纤维蛋白凝胶上的最佳接种方式及其生长情况。方法取角膜缘上皮组织块进行细胞培养。用针对鼠抗兔角蛋白3(CK3)的单克隆抗体AE5和抗增殖细胞核抗原(PCNA)抗体行间接免疫荧光鉴定。MTT法测定CLEC增殖活性。将传第二代的细胞分A、B两组分别接种到纤维蛋白凝胶表面和凝胶体内进行培养,在接种后5d、10d、15d时将凝胶溶解离心后计数细胞数,比较两种接种方式下细胞的增殖力。结果传第一、二代细胞培养3～5d后胞浆AE5染色阳性,胞核PCNA染色阳性,传第一代阳性细胞比例分别为31%、65%,传第二代阳性细胞比例分别为45%、79%。传第一代细胞在接种后6h内贴壁,1～2d时处于潜伏期,2～3d时处于快速生长期,3d时达85%以上汇合,细胞增殖活性最高,4d时细胞汇合达95%以上,增殖活性逐渐降低。接种后5d时A组细胞数低于B组,差异有统计学意义(P<0.05);10d和15d时A组细胞数均高于B组,差异均有统计学意义(均为P<0.05)。结论以纤维蛋白凝胶为载体培养CLEC时将细胞接种在凝胶表面比包埋在凝胶体内更有利于细胞增殖,适合用于CLEC移植培养的研究。

Objective To determine the optimum method of implanting rabbit corneal limbus epithelial cells （ CLEC） when using fibrin gel （ FG） as medium,and observe its growth.Methods The plants of limbus epithelium tissue were cultured in vitro.Expression of CK3 and PCNA in the cultured cells were indentified by indirect immunofluorescence staining.Proliferation activity of the cultured CLEC was measured by MTT.The second-generation CLEC were divided into group A and B,which were implanted respectively on the surface of FG and inside FG.At 5 days,10 days and 15 days after implantation,the gels were digested and removed by centrifugation to count the number of cells,then comparison of the proliferation activity was made between the two groups.Results The indirect immunofluorescence staining result indicated positive expression of CK3 in cytoplasm and PCNA in nucleus in first-generation and second-generation CLEC from 3 days to 5 days after cultivation,the proportion of positive cells in first-generation CLEC were 31% and 65%,in second-generation cells were 45% and 79%.The first-generation CLEC adhered to wall within 6 hours,were in potential stage from 1 day to 2 days,in rapid growth stage from 2 days to 3 days,joined to 85% at 3 days with the highest proliferation activity,then converged more than 95% at 4 days with gradually decreased proliferation activity.The cell number of group A at 5 days was smaller than that of group B（ P 0.05 ） ,at 10 days and 15 days group A were larger than group B（ both P 0.05） .Conclusion It is better to implant CLEC on the surface of FG than inside of it for higher proliferation activity.The former is fit for study on transplantation of CLEC.